由美国国立卫生研究院R01 HL74082，哈特韦尔基金会，聚焦超声手术基金会支持。

1。微泡生产<ol><li>为了准备白蛋白微泡（MBS），放置在一个烧瓶血清白蛋白的1％溶液与水相中的气毯以上（octafluoropropane）生理盐水。简言之超声配备一个扩展½“钛探头超声粉碎机（30秒）的解决方案，这一提法是Optison（GE Heathcare），这是在0.5 - 1.2 × 10 9 MBS /毫升的浓度范围内提供类似。确定平均MB与Multisizer库尔特计数器直径白蛋白MB的直径平均在这项研究中使用了1.93um ± 1.63um。 </li><li>编造脂质MBS，准备1 mg / ml的聚乙二醇- 40硬脂酸（Sigma公司，圣路易斯，密苏里州）和2毫克/毫升distearoyl磷脂（阿凡提极地血脂，雪花石膏，AL）的水性分散体和sonciate如以前（1.1）decafluorobutane气体。测定平均MB的直径与Multisizer库尔特计数器。在这项研究中所使用的脂质MB的平均直径为2.01um ± 1.29um。 </li></ol> 2。纳米粒子的制备<ol><li>这些方法适应的水在油中水乳化溶剂蒸发技术Davda（2002年）和Chappell（2008）所描述。 </li><li>准备在1000毫升的去离子（DI）水溶解的PVA20克2％的聚（乙烯醇）PVA溶液。允许不小的轰动板的解决方案，充分溶解过夜。离心10分钟，用0.​​22μm的无菌过滤器的过滤器在1000 RPM的解决方案，以消除任何残留不溶的聚乙烯醇。 </li><li>编造牛血清白蛋白（BSA）加载聚（乳酸 - 乙醇酸）（PLAGA）纳米粒子（NPS），溶于6毫升二氯甲烷（MC）的玻璃闪烁瓶180mg封顶85:15 PLAGA。旋涡2分钟MC / PLAGA解决方案。 </li><li>溶解于1.5毫升的PBS所需的有效载荷（牛血清白蛋白15毫克）。在两部分的PBS / BSA溶液加入间歇振荡的PLAGA / MC解决方案。 5分钟冰和解决方案，45W超声为120秒。 </li><li>添加MC / PLAGA /有效载荷/ PBS液的2％聚乙烯醇24毫升，在两个部分中间震荡。 </li><li>配售5分钟冰的解决方案和120超声在45W。 </li><li>搅拌12小时在通风柜的轰动板的NP乳液，让MC和NP稳定蒸发。 </li><li>离心20,000 rpm的暂停25分钟在4 ° C两次分离粒子，除去残留的聚乙烯醇。重悬在DI水和离心机8毫升沉淀10分钟在1000 RMP 4 ° C至隔离核动力源的100nm的人口。 </li><li>分离上清和闪光灯冻结在-80 ° C。冻干48小时冷冻样品。商店dessicator冻干颗粒于-80 ° C，直到使用时间。 </li></ol> 3。综合配送车辆制造VisEn化学债券议定书“（改编） <ol><li> VivoTag680转换羧基表面功能<ol><li>结合50μL1.0 M羟乙基，pH值7和丁二酸酐在25μL的DMSO（2.5毫克，25 ìmol）之一VivoTag680小瓶。这个解决方案，充分混合添加1.0 M NaOH溶液50μL;允许解决方案，在避光室温反应2小时。净化颗粒使用Bio - Rad公司BioGel（P100，中）与0.1 M的MES缓冲液，pH值6.0洗脱。收集的绿色带。 </li></ol></li><li>活化羧基改性VivoTag680 <ol><li>结合羧基改性VivoTag680在0.1 M的MES缓冲液，pH值6取得，与1毫克1 - 乙基-3 - （3 dimethylaminopropyl） - 碳二亚胺（EDC）和2.2毫克（N -羟基氨基NHS）。允许反应在室温下2小时。 </li><li>净化凝胶过滤多余的激活剂（EDC）激活颗粒，使用Bio - Rad公司BioGel（P100，中），用0.1 M的MES缓冲液，pH 6.0洗脱，收集的绿色带。立即共轭含胺分子（如牛血清白蛋白纳米微粒）的活性粒子。允许解决方案的反应在室温下2小时。胺共轭粒子在20000转离心60分钟，在4 ° C，浇注，离心后的上清液和0.1的MES缓冲resuspending从过剩的EDC纯化。 </li></ol></li><li>停在这里编造VivoTag680标记为给药研究纳米粒子。 </li><li>羧基改性的PLGA - BSA的VivoTag680纳米粒子（NP680）共轭白蛋白微泡<ol><li>结合NP680在0.1 M MES缓冲液，pH值6获得1毫克，1 - 乙基-3 - （3 dimethylaminopropyl）碳二亚胺（EDC）和N -羟基- sulfosuccinimide（磺酸基NHS）的2.2毫克。让溶液在室温下1小时反应。 </li><li>净化多余的EDC激活纳米粒子在20000转离心60分钟4 ° C，浇注，离心后的上清液。悬浮纳米粒子在0.1的MES缓冲液。 </li><li>洗白蛋白MBS在脱气PBS 3倍，从解决方案中删除多余的牛血清白蛋白。 </li><li>立即共轭含胺分子（如白蛋白微泡）的活性粒子。 NP680的解决方案，允许在室温下反应2小时。净化与脱气PBS洗涤三次未绑定NP680s NP680（MNCA）结合微泡。 </li><li>确定的使用库尔特计数器的MNCAs浓度。 </li></ol></li></ol> 4。肿瘤模型所有的动物实验与弗吉尼亚大学动物护理和使用委员会批准的动物协议的规定。 <ol><li> C6 Giloma大鼠肿瘤细胞株是由贾森的UVA博士希恩（夏洛茨维尔，弗吉尼亚州）提供。 <ol><li>细胞系进行了测试，以确保细胞被支原体免费。 </li></ol></li><li>保持细胞株中的F - 12K的营养补充混合16％马血清，3％胎牛血清（FBS），和1％的笔链球菌（GIBCO，美国），在37 ° C和5％的CO 2 。 </li><li>接种3 × 10 6 C6 Giloma肿瘤细胞在左后肢皮下注射300μL的PBS暂停C57BLJ6/Rag1小鼠（杰克逊）。让肿瘤为12天增长达到8 - 10MM，最大直径。 </li></ol> 在体内超声应用5。 <ol><li>麻醉小鼠腹腔（IP）组合注射盐酸氯胺酮（60毫克/公斤体重）和甲苯噻嗪（0.1毫克/公斤体重）治疗前。 </li><li>准备一个维护盐酸氯胺酮麻醉（20毫克/公斤体重）和甲苯噻嗪（0.3毫克/公斤体重）。管理作为需要。 </li><li> Cannulate每个动物的尾静脉注射，静脉注射（IV）的一个MB，MB / NP或MNCA解决方案管理。 </li><li>肿瘤灌注测量<ol><li>第四注入连续输液泵（2000年哈佛器械PHD;哈佛器械，Holliston，马）在15μl/min率脂质MB的解决方案（1 × 10 8 MBS / g体重0.3毫升0.9％生理盐水） 。 </li><li> ACUSON红杉512超声系统（西门子医疗解决方案，美国加州Mountain View）8月13 MHz的线性15L8探头配备使用在这项研究中，量化的对比增强肿瘤灌注。夫妻15L8探头肿瘤的水基超声凝胶（帕克实验室Aquasonic 100;帕克实验室公司，费尔菲尔德，新泽西州）。扫描肿瘤在B模式，以获得最佳的成像平面。 </li><li>在对比脉冲序列（CPS）模式，获得一个连续的视频捕获微泡信号强度前5秒，20以下，高振幅的“突发”的13Hz的帧速率的脉冲。在四个平面​​成像重复的测量。 </li><li>等待10分钟，注入白蛋白MBS和启动治疗的低频率的治疗，以使血脂MBS清除流通。 </li></ol></li><li>治疗性超声治疗<ol><li> 10分钟以下的肿瘤灌注测量（5.3）夫妇0.75英寸直径为1 MHz的重点不​​突出传感器（A314S;的Panametrics，沃尔瑟姆，马）以上的侧翼肿瘤的皮肤。四，注入白蛋白MB（1 × 10 5 MBS / g体重0.3毫升0.9％生理盐水），MB / NP（1 × 10 5 MBS /克和0.2微克粒子/克体重0.3毫升0.9％生理盐水）或MNCA解决方案（1 × 10 5 MNCAs /克体重0.3毫升0.9％生理盐水）。 </li><li>药物输送治疗<ol><li> Insonate为60分钟的“1爆裂”脉冲序列在连续输注的粒子，MBS和NPS的合作注入，或MNCAs（5.4.1）（5.4.2.2）。 </li><li> “1爆”脉冲序列由连续100 1 MHz的正弦信号（占空比= 0.00002），1V峰到峰值振幅波形发生器（AFG - 310;泰克公司日前宣布，比弗顿，或）。放大55分贝（埃尼集团3100LA电子导航行业，理查森，TX）与功率放大器的RF波形信号。 </li></ol></li><li>烧蚀治疗<ol><li>相同5.4.2.1，但是与“突发中等”或​​“5连拍 - 扩展”insonate脉冲序列</li><li> “5突发中的”脉冲序列由5000 1 MHz的正弦波（占空比= 0.005）1V峰 - 峰幅度。 “5突发Exteneded”脉冲序列由10000 1 MHz的正弦波（占空比= 0.01）1V峰 - 峰幅度。 </li><li>在插入一根针进入肿瘤的热电偶探头（欧米茄T型，HYP1-30-1/2-TG-60-SMPW-M）2厘米实验监测肿瘤温度的时间当然。记录温度测量，每五分钟。 </li></ol></li></ol></li><li>重复肿瘤灌注测量5.3 10分钟以下治疗服务。 </li></ol> 6。肿瘤灌注的定量<ol><li>红杉上使用的CPS程序，输入ACQ模式。选择日E区包括完整的肿瘤体积的利益。灌注复苏曲线的形式会产生Y = A（1 - E -βt）+（Sadlowski 2002年，2001年色度;叶2004年）是适合的CPS数据。评估β，一个红血细胞的速度，前处理和后处理的相对措施。 </li><li>出口CPS的文件进行离线分析。数据转换成AVI文件。打破AVI文件转换成图像序列帧一帧。使用康柏的Visual Fortan，或类似的软件包，生成时间平均从破坏性脉冲的所有帧图像为8秒以下的破坏性脉冲。 </li><li>要确定肿瘤灌注区，使用ImageJ软件，应用门槛247 8叮咬的时间平均CPS的形象和量化增强像素内的肿瘤体积的百分之。相应的B型图像覆盖的时间平均阈值的图像，准确地确定肿瘤的面积。 </li><li> ％的平均灌注获得在给定时间点为特定肿瘤的最后的灌注区的面积至少四个郊野公园段剪辑。 </li><li>以％灌流区（6.4）和β（0.1），以确定相对肿瘤血流的产品。 </li></ol> 7。纳米粒子在肿瘤的生物分布<ol><li> 12天前与荧光标记的低alfasprot饮食（哈伦，印第安纳波利斯）核动力源小鼠治疗，以减少正常小鼠议员成像时造成的自体荧光。 </li><li>剃须成像网站（侧翼肝）删除所有的头发。 </li><li>删除与化学脱毛剂，如奈尔，残留的头发。 <ol><li>所有剩余的化学头发卸妆冲洗或可能发生的化学品燃烧。 </li></ol></li><li>如在5.4.2中所述的治疗，治疗后0，1，4，和24小时前（FMT系统VisEn医疗）的图像小鼠。 </li><li>成像与盐酸氯胺酮（60毫克/公斤体重）和甲苯噻嗪（0.1毫克/公斤体重）的IP组合注射麻醉小鼠之前。将鼠标上的成像盒。收购白色的光反射和荧光模式图像。 VT680渠道开展荧光层析成像。 </li><li>采用的裂变材料条约的软件来生成三维重建成像数据利用归一出生方程。重建后，选择利益卷（VOI），在所有3个成像平面（X，Y，Z）的绘图区域的利益率（ROI）。平均荧光值的总VOI体积和荧光浓度，由于荧光标记的BSA的纳米粒子，会产生VOI的无所不包的脚和肝脏。 </li></ol> 8。肿瘤的生长速度<ol><li>评估采取日常使用数字口径测量肿瘤体积。计算椭球近似的肿瘤体积V = 1 / 6πABC。其中A，B和C三个正交平面测量肿瘤的最大直径。 </li></ol> 9。肿瘤的处理和分析<ol><li> 7天以下治疗安乐死动物。 Cannulate10毫升2％的左心室和抽血血液肝素化三氯化钙缓冲液（0.68毫米）灌注10ml三氯化钙中2缓冲液（0.68毫米），每10分钟在100毫米汞柱。 </li><li>灌注修复组织10分钟灌注4％多聚甲醛的PBS（4℃），在100毫米汞柱。允许样品固定为60分钟。 </li><li>海关的标本，嵌入石蜡，切成5微米的部分。 </li><li>执行标准的苏木精伊红染色，以评价超声暴露的结果，可能发生的病理变化。 </li><li>检测细胞凋亡，使用终端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法（TUNEL）法（ApoptTag套件，InterGen公司有限公司，诺克罗斯，GA，美国）。 </li></ol> 10。代表性的成果 1。纳米粒子的制备（2.0） <ol><li>如果这个协议是正确的预制的粒子，将是球形的形状，通过扫描电子microcopy（SEM）确定，并有大约100nm的高斯分布，光散射技术决定。代表结果如图1所示。 </li></ol> 2。靶向给药（5.4.1） <ol><li>根据发表的研究报告在我们的实验室（宋2002年，查普尔2​​008），在图2中，超声破坏微泡造影剂在肿瘤组织血管纳米粒子增强交付结果未公布结果进行。我们还表明，我们的MNCA交付技术成果在提高纳米粒子交付紧随治疗。 </li></ol> 3。肿瘤消融（5.4.3） <ol><li>我们已经表明在相对较低的占空比，长时间超声微泡的破坏，（0.005-0.01），在机械消融结果肿瘤生长的肿瘤微血管和回归。如果这个协议是正确执行，不要指望在变化（I）肿瘤的生长率（图3）;（二）肿瘤的血流动力学变化（图4），（三）坏死区;及（iv）凋亡。 </li></ol><img src="/files/ftp_upload/2145/2145fig1.jpg" alt="图1" /> 图1。PLAGA表征轴承牛血清白蛋白。 （一）正确装配纳米粒子的SEM图像。 （二）不当编造的纳米粒子的SEM图像。 <img src="/files/ftp_upload/2145/2145fig2.jpg" alt="图2" /> （一）介导的荧光断层成像（FMT）的图像的超声治疗（顶部）和控制处理（下）皮下神经胶质瘤的治疗后，立即用一个MNCAs，纳米粒子（NPS）轴承680荧光信号叠加在图2。灰度平面激发光的形象。 <img src="/files/ftp_upload/2145/2145fig3.jpg" alt="图3" /> 图3。以下五爆裂延长脉冲协议的微泡insonation代表在肿瘤生长的倍数变化。 <img src="/files/ftp_upload/2145/2145fig4.jpg" alt="图4" /> 图4。B型（A，C）和超声造影（B，D）在小鼠皮下C6胶质瘤肿瘤的影像。在最初的预处理图像（一）多为低回声肿瘤的边界一直追溯到蓝色。 （二）预处理时间平均提高静脉注射造影剂后显示。治疗后，注射造影剂前，肿瘤又大多是低回声（四）。注射造影剂后，有明显减少，在肿瘤的增强。

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mean+Flow+Rate+and+Intergrated+Perfusion+Estimates+Obtained+with+Contrast-Assisted+Ultrasound.">Mean Flow Rate and Intergrated Perfusion Estimates Obtained with Contrast-Assisted Ultrasound.</a> IEEE Ultrasonics Symposium. , 1977-1980 (2002).</li><li>Song, J., Chappell, J. C., Qi, M., VanGieson, E. J., Kaul, S., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+injection+site,+microvascular+pressure+and+ultrasound+variables+on+microbubble-mediated+delivery+of+microspheres+to+muscle.">Influence of injection site, microvascular pressure and ultrasound variables on microbubble-mediated delivery of microspheres to muscle.</a> J Am Coll. Cardiol. 39, 726-731 (2002).</li><li>Sheikov, N., McDannold, N., Vykhodtseva, N., Jolesz, F., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+blood-brain+barrier+opeing+induced+by+ultrasound+in+the+presences+of+microbubbles.">Cellular mechanisms of blood-brain barrier opeing induced by ultrasound in the presences of microbubbles.</a> Ultrasound Med. Biol. 30, 979-989 (2004).</li><li>Tanter, J., Fink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+and+steering+through+absorbing+and+aberrating+layers:+Application+to+ultrasonic+propagation+through+the+skull.">Focusing and steering through absorbing and aberrating layers: Application to ultrasonic propagation through the skull.</a> J Acoust Soc Am. 103, 2403-2410 (1998).</li><li>Yeh, C. K., Kruse, D. E., Lim, M. C., Redline, D. E., Ferrara, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+High+Frequency+Destruction/Reperfusion+System.">A New High Frequency Destruction/Reperfusion System.</a> IEEE. 1, 433-436 (2003).</li></ol>

关键步骤小鼠尾静脉插管的： 静脉注射入小鼠尾静脉，可以是一个具有挑战性的的过程。然而，尾静脉导管，可以大大提高了一个成功注入的可能性。为了使导管，来回反复弯曲25号针头，直到它打破从枢纽。钝端插入体育20月底油管和密封胶与硅的连接。要准备的导管插管，将装有1％肝素生理盐水的凯瑟注射器和液体的死腔的导管注入。尾静脉麻醉鼠标在?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>ApoptTag kit</td>
<td>&nbsp;</td>
<td>Intergen Co.</td>
<td>S7110</td>
<td>&nbsp;</td>
<tr>
<td>un-capped 85:15 poly(lactic-co-glycolic acid) (PLAGA)</td>
<td>&nbsp;</td>
<td>Lakeshore Biomaterials</td>
<td>Custom</td>
<td>&nbsp;</td>
<tr>
<td>Vivo Tag 680</td>
<td>&nbsp;</td>
<td>VisEn Medical</td>
<td>10120</td>
<td>Used to Tag BSA</td>
</tr>
<tr>
<td>Poly(vinyl alcohol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>363136</td>
<td>&nbsp;</td>
<tr>
<td>MicroTip Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>S-4000</td>
<td>&nbsp;</td>
<tr>
<td>Sequoia</td>
<td>&nbsp;</td>
<td>Simons Medical</td>
<td>P.O.A</td>
<td>Equipped with CPS </td>
</tr>
<tr>
<td>FreeZone 2.5</td>
<td>&nbsp;</td>
<td>Labconco</td>
<td>7670020</td>
<td>Equipped with Nitrogen Trap</td>
</tr>
<tr>
<td>Methylene chloride (CH2Cl2)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D37-500</td>
<td>&nbsp;</td>
<tr>
<td>FMT 250</td>
<td>&nbsp;</td>
<td>VisEn Medical</td>
<td>P.O.A</td>
<td>&nbsp;</td>
<tr>
<td>F-12K Nutrient Mixture</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21127-022</td>
<td>&nbsp;</td>
<tr>
<td>polyethyleneglycol-40 stearate </td>
<td>&nbsp;</td>
<td>Sigma Chemical </td>
<td>9004-99-3</td>
<td>&nbsp;</td>
<tr>
<td>distearoyl phosphatidylcholine</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>770365</td>
<td>&nbsp;</td>
<tr>
<td>Multisizer Coulter Counter</td>
<td>&nbsp;</td>
<td>Beckman Coulter </td>
<td>P.O.A</td>
<td>&nbsp;</td>
<tr>
<td>Waveform Generator </td>
<td>&nbsp;</td>
<td>Tektronix, Inc.</td>
<td>AFG-310</td>
<td>&nbsp;</td>
<tr>
<td>water-based ultrasound gel </td>
<td>&nbsp;</td>
<td>Parker Laboratories</td>
<td>Aquasonic 100</td>
<td>&nbsp;</td>
<tr>
<td>Infusion pump </td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Harvard Apparatus PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) </td>
<td>&nbsp;</td>
<td>Pierce Biotechnology</td>
<td>25952-53-8</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysulfosuccinimide (Sulfo-NHS)</td>
<td>&nbsp;</td>
<td>Pierce Biotechnology</td>
<td>106627-54-7</td>
<td>&nbsp;</td>
<tr>
<td>Succinic anhydride</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>603902</td>
<td>&nbsp;</td>
<tr>
<td>Power Amplifier </td>
<td>&nbsp;</td>
<td>Electronic Navigation Industries</td>
<td>ENI 3100LA</td>
<td>&nbsp;</td>
<tr>
<td>Needle Thermocouple Probe </td>
<td>&nbsp;</td>
<td>Omega</td>
<td>HYP1-30-1/2-T-G-60-SMPW-M</td>
<td>&nbsp;</td>
<tr>
<td>BioGel (P100, medium)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>150-4170</td>
<td>&nbsp;</td>
<tr>
<td>.75&#x2019;&#x2019; diameter 1 MHz unfocused transducer</td>
<td>&nbsp;</td>
<td>Panametrics</td>
<td>A314S </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

contrast ultrasound targeted treatment of gliomas in mice via drug-bearing nanoparticle delivery and microvascular ablation

我们正在开发的微创造影剂微泡造影剂的基础微血管的通透性和/或消融是由不同的超声脉冲参数控制的治疗方法。具体来说，我们正在测试这种办法是否可用于药物输送和微血管消融治疗恶性脑肿瘤。已经进行初步研究，以确定是否有针对性的药物轴承纳米粒子交付，可以通过100nm的聚（乳酸 - 乙醇酸）（PLAGA）坚持白蛋白炮轰微泡的纳米粒子组成的“复合”投递代理的超声导破坏促进。我们这些代理商表示微气泡纳米粒子复合代理人（MNCAs）。当与超声定位皮下C6脑胶质瘤，我们观察到纳米粒子交付立即与与纳米粒子和8.5倍的增长比非治疗肿瘤共同管理的微泡治疗肿瘤在MNCA治疗肿瘤中增加了4.6倍。此外，在许多癌症的应用，我们认为它可能是可取的执行结合消融肿瘤微循环，这将导致肿瘤的缺氧和凋亡的靶向给药的。为此，我们已经测试非theramal气蚀引起的微血管消融的疗效，表明这种方法引出肿瘤灌注减少，细胞凋亡，显着的生长抑制作用，并坏死。两者合计，这些结果表明，超声定位的方法有可能增加肿瘤坏死创建通过微血管的消融和/或同时提高药物的有效载荷在胶质瘤治疗的效率。

Watch this Scientific Journal Video about Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation at JoVE.com

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

微泡Insonation是很有前途的战略肿瘤消融时间平均减少声学权力，以及为靶向给药疗法。本研究的目的是开发低占空比超声脉冲战略和nanocarriers最大限度地发挥非热微血管消融和有效载荷运送到皮下C6脑胶质瘤。

超声造影有针对性的治疗胶质瘤的小鼠通过药物轴承纳米粒子交付和微血管消融

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

contrast-ultrasound-targeted-treatment-gliomas-mice-via-drug-bearing

Research

JoVE Journal

Bioengineering

15.8K 次观看.  University of Virginia. 微泡Insonation是很有前途的战略肿瘤消融时间平均减少声学权力，以及为靶向给药疗法。本研究的目的是开发低占空比超声脉冲战略和nanocarriers最大限度地发挥非热微血管消融和有效载荷运送到皮下C6脑胶质瘤。

视频: Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

有针对性的超声微泡销毁技术简介

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

高强度的实时监控聚焦超声（HIFU）消融<em&gt;体外</em&gt;使用谐运动成像的聚焦超声犬肝脏（HMIFU）

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

用三种互补方法综合评价胎盘靶向药物的有效性和安全性

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

患者特异性聚乙烯酒精幻影制造与超声波和X射线对比脑肿瘤手术规划

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...